(a) Field of the Invention
The invention relates to a new “gene-switch” (cumate-inducible switch) for mammalian cells. This switch is as useful in the development of expression systems and cell-based assays for functional genomics as in the generation of viral vectors for gene therapy.
(b) Description of Prior Art
Tightly controlled inducible expression of foreign proteins would greatly aid functional studies in heterologous systems. The ability to regulate both the level and the duration of expression would allow the study of proteins whose constitutive expression might not be tolerated by the cell. A number of inducible systems endogenous to mammalian cells involving regulation by heavy-metals (Brinster, R. L., et al. Nature (London) 296: 39-42, 1982; Mayo, E. K., et al. Cell 29: 99-108, 1982; and Searle, P. F., et al. Molecular and Cellular Biology 5: 1480-1489, 1985), steroid hormones (Hynes, N. E., N. Kennedy, et al. Proc. Natl. Acad. Sci. USA 78: 2038-2042, 1981; Lee, F., et al. Nature (London) 294: 228-232, 1981; and Klock, G., et al. Nature (London) 329: 734-736, 1987), heat shock ((Nouer, L. p.-., Heat Shock Response. Boca Raton, Fla., Ed. CRC, 1991) (reviewed in Mullick, A. and B. Massie Encyclopedia of Cell Technology pp. 1140-1164, 2000)) are widely used. However, a major limitation of these inducible mammalian promoters is the pleitropic effects of the inducers (heat shock, glucocorticoids etc.).
To overcome these problems, prokaryotic (Gossen, M., et al. TIBS 18: 471475, 1993) and insect regulatory systems (No, D., et al. Proc. Natl. Aced. Sci. USA 93: 3346-3351, 1996) have been adapted to construct gene switches that function in mammalian cells. Since inducer molecules are not expected to have targets in mammalian cells, the possibility of interference with cellular processes is reduced.
Of the prokaryotic proteins, two have proved particularly useful, the repressors from the lac (Brown, M., et at. Cell 49: 603-612, 1987; and Hu, M. C. -T. and N. Davidson Cell 48: 555-566, 1987) and the tet operons (Blau, H. M. and F. M. V. Rossi, Proc. Natl. acad. sci. USA 96: 797-799, 1999). Both have been incorporated in eukaryotic inducible expression systems using different strategies to control activation and repression of expression. Activation of expression is mediated by a chimaeric transactivator protein formed by the fusion of the bacterial repressor with an activation domain (Gossen, M. and H. Bujard, Proc. Natl. acad. sci. USA 89: 5547-5551, 1992, and Gossen, M., et al. Science 268: 1766-1769, 1995). The transactivator is able to activate transcription when bound to its DNA recognition sequence placed upstream of the minimal promoter. The ability of the activator to bind DNA is dependent on the presence/absence of the inducer molecule. Repression of expression is mediated by the repressor bound to operator sites placed downstream of the minimal promoter in the absence of inducer and repression is relieved on the addition of the inducer (Brown, M., et al. Cell 49: 603-612, 1987).
It would be highly desirable to be provided with an alternate activation/repression switch for expression of eukaryotic proteins.